Morphology and wettability of dielectric layers are crucial properties that affect the electrowetting-on-dielectric (EWOD) performance of a working liquid. In this work, the use of a poly (vinyl chloride)(PVC) microfiber-modified PVC dielectric layer as an electrowetting-on-dielectric (EWOD) substrate is explored. Imaging with scanning electron and atomic force microscopy revealed intertwined microfibers on the original PVC film after PVC deposition via electrospinning. Hydrophobicity of the PVC layer was enhanced by the presence of microfibers, with the contact angle (CA) for a water droplet increasing from 84.9 to 123.9. EWOD behavior of various liquids on the microfiber-modified PVC layer was investigated within a DC voltage range of 0 to 200 V DC. Gold nanofluid exhibited the largest change in CA of 57, while smaller changes were observed for KOH (19.6), KCl (21.2), and water (21). A mechanism explaining the observed reduction in contact angle for a gold nanofluid droplet is presented. Our results suggest the promising potential of PVC film-PVC microfiber as a hydrophobic dielectric material for EWOD applications involving diverse liquids, including gold nanofluids.